Aryl hydrocarbon receptor (AHR) is a potential tumour suppressor in pituitary adenomas

Abstract

Pituitary adenomas (PA) represent the largest group of intracranial neoplasms and yet the molecular mechanisms driving this disease remain largely unknown. The aim of this study was to use a high-throughput screening method to identify molecular pathways that may be playing a significant and consistent role in PA. RNA profiling using microarrays on eight local PAs identified the aryl hydrocarbon receptor (AHR) signalling pathway as a key canonical pathway downregulated in all PA types. This was confirmed by real-time PCR in 31 tumours. The AHR has been shown to regulate cell cycle progression in various cell types; however, its role in pituitary tissue has never been investigated. In order to validate the role of AHR in PA behaviour, further functional studies were undertaken. Over-expression of AHR in GH3 cells revealed a tumour suppressor potential independent of exogenous ligand activation by benzo α-pyrene (BαP). Cell cycle analysis and quantitative PCR of cell cycle regulator genes revealed that both unstimulated and BαP-stimulated AHR reduced E2F-driven transcription and altered expression of cell cycle regulator genes, thus increasing the percentage of cells in G₀/G₁ phase and slowing the proliferation rate of GH3 cells. Co-immunoprecipitation confirmed the interaction between AHR and retinoblastoma (Rb1) protein supporting this as a functional mechanism for the observed reduction. Endogenous Ahr reduction using silencing RNA confirmed the tumour suppressive function of the Ahr. These data support a mechanistic pathway for the putative tumour suppressive role of AHR specifically in PA, possibly through its role as a cell cycle co-regulator, even in the absence of exogenous ligands.

Keywords: AHR; microarray; pituitary adenoma; tumour suppressor.

Figure 1

qPCR of target genes to confirm data obtained by microarray analysis. Genes with de-regulated expression as compared to normal pituitary were analyzed using GAPDH and EMC7 as housekeeping genes. (CYP1B1, cytochrome P450 1B1, GST; gluthionine S-transferase, ALDH3A1; aldehyde dehydrogenase family 3, A1, JUN; Jun oncogene, NFPA; non-functioning PA, FPA; functional PA, GH-S; growth hormone secreting PA, PRL-S; prolactin-secreting PA, ACTH-S, adrenocorticotropic hormone-secreting PA). qPCR experiments were done in triplicate and error bars indicate standard error. (*P<0.05).

Figure 2

Proliferation (MTT) assays were carried out to study the role of AHR signaling on GH3 cell proliferation at different time points. (A) GH3 cells treated with BαP at different concentrations over three days. (B) GH3 cells transfected with increasing amounts of AHR/EV (pcDNA3) expression vectors and analyzed at three different time points from transfection. Insert shows increase in expression of transfected AHR with western blot using B-actin as loading control (C) GH3 cells transfected with AHR or EV plasmid and treated with BαP at different concentrations 24 hours after transfection and assayed at different time points post-transfection. (D) GH3 cells were transfected with Ahr or Control (Ctrl) siRNA at different concentrations and proliferation measurements taken at 3 time points. Insert shows a western blot reflecting the efficiency of Ahr silencing using siRNA. Experiments were done in repeats of 5 per treatment and each experiment was repeated at least three times. (*P< 0.05, error bars represent standard error).

Figure 3

Co-immunoprecipitation of Ahr and Rb1 in Gh3 cells and the effect of AHR over-expression and exogenous stimulation on cell cycle regulators in GH3 cells. (A) Co-IP was carried out in GH3 cells and western blot with 20μg of cell lysate or precipitated proteins was run using anti-Rb1 and ant-Ahr antibodies on a negative control with lysate run through Ig beads with no antibody (Ctrl), the whole cell lysate prior to precipitation (input) and Co-IP using anti-AHR for pull down. Lane 1 of the co-IP represents untreated cells, lane 2 contains the precipitate from GH3 cells over-expression AHR, lane 3 from GH3 cells treated with 1µM BαP prior to lysis and lane 4 with lysate from GH3 cells with knocked down Ahr protein. (B) GH3 cells were co-transfected with AHR or EV together with an E2F-driven luciferase reporter plasmid and treated with no BαP (-), 100nM or 1µM BαP for 24 hours prior to lysis and luciferase readings. For knock down experiments, GH3 cells were transfected with reporter plasmid and Ahr or control (Ctrl) siRNA) for 24 hours prior to luciferase reading. (C) GH3 cells were transfected with either AHR/EV or Ahr siRNA and treated with 1μM BαP for 24 hours prior to lysis and RNA extraction. Experiments were repeated three times and luciferase studies were done in triplicate while qPCR readings were done in duplicate and repeated three times. (*P<0.05, **P< 0.01, error bars indicate standard error).

Figure 4

Gene expression analysis of specific cell cycle regulator genes in 31 pituitary adenomas, divided by tumour type and shown as fold changes over control pituitary RNA. qPCR readings were done in duplicate and repeated three times. (*P<0.05, error bars indicate standard error).

Figure 5

Cell cycle analysis was carried out on GH3 cells transfected with AHR/EV or Ahr/Ctrl siRNA and treated with vehicle or BαP for 24 hours prior to fixation and flow cytometry. (A) Percentage of GH3 cells in G0/G1 phase in differently treated cells. Cell cycle analysis was done in triplicate and experiment was repeated three times using synchronized cells. (B) Cell Quest Pro software was used to gate the cell population and only gated cells were used for the final cell cycle analysis. Areas designated as belonging to G0/G1 (M1), S phase (M2) and G2 phase were assigned as shown. (*P<0.05, error bars represent standard error).

Figure 6

Illustration of AHR activity in pituitary cells. The AHR may act through two pathways in the pituitary cells which can either be distinct in activity or influence each other by sequestering AHR protein. Pathway 1 illustrates the standard canonical xenobiotic signaling by AHR which is activated by exogenous or endogenous ligands to translocate to the nucleus together with ARNT and transcribe target genes such as CYP1A1, AHR among others. The second pathway involves the influence of AHR on cell cycle regulators. Through its interaction with Rb protein, AHR is able to maintain it in its hypo-phosphorylated state where it inhibits E2F activity and hence represses cell cycle progression. Alternatively, AHR may also increase expression of CDKN1A and CDKN1B genes resulting in increased p21 and p27 protein levels which also repress cyclin/CDK activity and maintain the hypo-phosphorylated state of Rb, thereby also reducing cell cycle progression.